Verification Protocol Guide Accu Shot... · 2. The United States Pharmacopeia‐National Formulary....

Verification Protocol Guide

MicroBioLogics® Verification Protocol Guide

TIB.281 REV.2010.FEB.04 Page 2 of 17

Table of Contents

1. Introduction 31.1 Safety Guidelines 41.2 Available Technical Information 41.3 References 4Acknowledgments 5

2. Verification Plan 52.1 Intended Location of Use 52.2 Microorganisms to be Tested 52.3 Scope of Verification 62.4 Verification Tests 62.5 Acceptance Criteria 72.6 Technician (s) Performing Verification 72.7 Approval of Plan 8

3. Verification of Traceability, Counts and Purity 83.1 Materials Needed for Verification 83.2 Medium Information 93.3. Documentation of Traceability to Reference Culture 93.4 Counts and Purity 103.5 Stability 113.6 Macroscopic and Microscopic Characteristics 133.7 Biochemical and/or Molecular Identity (If Required) 15

4. Summary and Conclusion 154.1 Summary of Data 154.2 Comments 164.3 Conclusion 174.4 Review and Approval 17



1. Introduction

The purpose of this Verification Guide is to help customers smoothly and efficiently verify that EZ‐Accu Shot™ microorganism strains are suitable for use in the Growth Promotion Test described in the United States Pharmacopeia (USP), the European Pharmacopoeia (EP), and the Japanese Pharmacopeias (JP). EZ‐Accu Shot™ microorganisms are lyophilized, standardized, stable suspensions that deliver less than 100 CFU (colony forming units) when used as directed. Once verified, EZ‐Accu Shot™ microorganism strains can be used as an easy and precise alternative to the seed‐lot system and the time‐consuming process of inoculum preparation.

EZ‐Accu Shot™ microorganism strains meet the following pharmacopeial requirements: 1. Each strain delivers less than 100 CFU per 0.1 mL when used as directed. 2. Each strain is derived from a reference culture strain recommended by the harmonized pharmacopeias

for growth promotion testing. The strains are traceable to the American Type Culture Collection (ATCC®). MicroBioLogics® is licensed by ATCC® to sell derivatives of ATCC® original microorganism strains.

3. Each strain is less than five passages from reference culture (the original master strain). 4. The identity of each strain has been confirmed by MicroBioLogics™.

In addition to meeting pharmacopeial requirements, EZ‐Accu Shot™ microorganisms have the following benefits: 1. Lyophilized microorganism preparations dissolve in seconds. 2. There is little preparation time. 3. Microorganism suspensions can be used for eight hours if refrigerated after rehydration. 4. A Certificate of Assay is provided with every lot. The certificate lists the mean assay value (CFU

concentration) obtained at MicroBioLogics®. 5. A Certificate of Analysis can be downloaded for every lot from the MicroBioLogics® website. The

certificate lists such information as reference number, number of passages from reference culture, and macroscopic, microscopic and phenotypic characteristics when appropriate.

The Verification Guide has been divided into three parts. Part 1: Verification Plan

• The laboratory determines the scope of the verification.

• The laboratory determines acceptance criteria.

• The plan is approved by management. Part 2: The laboratory verifies the following for each EZ‐Accu Shot™ strain:

• Traceability. Strains should be traceable to an original reference culture strain recommended by the pharmacopeias and should be less than five passages from the original master strain.

• Counts. Counts should be less than 100 CFU per agar plate, when product is used as directed.

• Purity. Contamination should not be seen on agar plates inoculated with the EZ‐Accu Shot product.

• Stability. Microorganism suspensions should be stable for 8 hours after pellets are hydrated. Laboratories do not have to test stability if they do not intend to refrigerate microorganism suspensions.

• Identity. Identification methods can range from confirming microscopic and macroscopic microorganism characteristics to performing biochemical or molecular tests.

Part 3: Verification Summary

• The laboratory summarizes the data and determines if EZ‐Accu Shot™ strains meets acceptance criteria.

• The laboratory submits the Verification Protocol to management for approval.



1.1 Safety Guidelines EZ‐Accu Shot™ preparations contain potentially harmful microorganisms. All microorganisms produced by MicroBioLogics® are BioSafety Level 1 or 2. Guidelines for handling microorganisms can be found on the MicroBioLogics website in a Technical Bulletin called “Microorganism Biosafety Levels”. Directions for handling a microorganism spill can be found in the EZ‐Accu Shot™ product insert. Before using the EZ‐Accu Shot™ product the MSDS should be downloaded and filed electronically or on site. There are two MSDS sheets available for the product. One MSDS is for Lyophilized Microorganism Preparations and the other MSDS is for Microorganism Hydrating Fluid.

1.2 Available Technical Information The following documents are available to help with your validation.

• MSDS for Lyophilized Microorganism Preparations, MSDS.244

• MSDS for Microorganism Hydrating Fluid, MSDS.208

• “Microorganism Biosafety Levels”, TIB.072

• EZ‐ Accu Shot™ Product Insert, PI.276

• EZ‐Accu Shot™ Illustrated Instructions, LIT. 280

• “Growth Promotion Test Guide for EZ‐CFU™, EZ‐CFU One Step™ and EZ‐Accu Shot™”, TIB.264

• ATCC® 16404™ Name Change Letter, LIT.256 The Name Change Letter explains why the name of ATCC® 16404™ was changed to Aspergillus brasiliensis.

Documents are available at www.microbiologics.com. An easy way to obtain the documents is to use the search box. Search for documents by entering key words such as MSDS or biosafety.

1.3 References The references used to create this guide are listed below. 1. The United States Pharmacopeia‐National Formulary. 2009. <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests, First Supplement to USP32‐NF27 2. The United States Pharmacopeia‐National Formulary. 2009. <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms, First Supplement to USP32‐NF27 3. The United States Pharmacopeia‐National Formulary. 2009. <71> Sterility Tests, First Supplement to USP32‐NF27 4. The United States Pharmacopeia‐National Formulary. 2009. <1226> Verification of Compendial Procedures FDA U.S. Food and Drug Administration. CFR ‐ Code of Federal Regulations Title 21. 2009‐04‐01. Laboratory Records 21 CFR 211.194(a)(2) 5. The United States Pharmacopeia‐National Formulary. 2009. <1111> Microbiological Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use 6. European Pharmacopoeia. 2009. 2.6.12, Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests.



7. European Pharmacopoeia. 2009. 2.6.13, Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms. 8. European Pharmacopoeia. 2009. 2.6.1, Sterility Tests. 9. Japanese Pharmacopoeia. May.19. 2009. 4.05, Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests and Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms. 15th Partial Revision (English version) 10. Japanese Pharmacopoeia. May. 19. 2009. 4.06, Sterility Tests. 15th Partial Revision (English version)

Acknowledgements

The ATCC Licensed Derivative Emblem, the ATCC Licensed Derivative word mark and the ATCC catalog marks are trademarks of ATCC. MicroBioLogics, Inc. is licensed to use these trademarks and to sell products derived from ATCC® cultures 2. Verification Plan

2.1 Intended Location of Use Company Name: __________________________________________________________________________

Address: __________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

Department: __________________________________________________________________________

Signature: _______________________________________________________ Date: ___________

2.2 Microorganisms to be Tested In Table 1, check (✔) EZ‐AccuShot™ microorganisms included in the verification study.

Table 1: EZ‐Accu Shot™ Microorganisms

Microorganism ATCC® number EZ‐Accu Shot™ Product number

Check (✔)if included in study

Aspergillus brasiliensis ATCC® 16404™* 0392A

Bacillus subtilis subsp. spizizenii ATCC® 6633™* 0486A

Candida albicans ATCC® 10231™* 0443A

Clostridium sporogenes ATCC® 19404™* 0317A

Clostridium sporogenes ATCC® 11437™* 0487A

Escherichia coli ATCC® 8739™* 0483A

Pseudomonas aeruginosa ATCC® 9027™* 0484A

Staphylococcus aureus subsp. aureus ATCC® 6538™* 0485A



2.3 Scope of Verification Briefly summarize the purpose of the Verification.

2.4 Verification Tests List the Tests which the laboratory will perform in order to verify the EZ‐Accu Shot™ product.



2.5 Acceptance Criteria Describe acceptance criteria for determining suitability of EZ‐Accu Shot™ microorganisms.

2.6 Technician(s) Performing Verification Name and signature of technician(s) performing validation.

Name (print) _______________________________________________Title____________________________

Signature__________________________________________________Date____________________________

Department________________________________________________________________________________

Company Name ____________________________________________________________________________

Address ___________________________________________________________________________________

Name (print) _______________________________________________Title____________________________

Signature__________________________________________________Date____________________________

Department________________________________________________________________________________

Company Name ____________________________________________________________________________

Address ___________________________________________________________________________________

Name (print) _______________________________________________Title_____________________________

Signature__________________________________________________Date_____________________________

Department_________________________________________________________________________________

Company Name _____________________________________________________________________________

Address ___________________________________________________________________________________



Name (print) _______________________________________________Title_____________________________

Signature__________________________________________________Date_____________________________

Department_________________________________________________________________________________

Company Name _____________________________________________________________________________

Address ____________________________________________________________________________________

2.7 Approval of Plan Signature(s) of management staff

Name (print)_______________________________________________Title_____________________________

Signature__________________________________________________Date____________________________

Name (print)_______________________________________________Title_____________________________

Signature__________________________________________________Date____________________________

Name (print)_______________________________________________Title_____________________________

Signature__________________________________________________Date____________________________

Name (print)_______________________________________________Title_____________________________

Signature__________________________________________________Date____________________________

3. Verification of Traceability, Counts, and Purity

3.1 Materials Needed for Verification 3.1.1 The following materials are needed for testing counts and purity.

EZ‐Accu Shot™ lyophilized microorganism preparations Hydrating Fluid (as received with each EZ‐Accu Shot™ kit) Calibrated pipetter and sterile tips Media such as Soybean Casein Agar (Tryptic Soy Agar) and Sabouraud Dextrose Agar Vortex Mixer Sterile Spreaders Incubator at 20‐25°C Incubator at 30‐35°C

Glass slides Gram Stain Kit for staining bacteria Lactophenol Blue Stain and clear tape for staining Aspergillus brasiliensis

3.1.2 Macroscopic characteristics should match the description of the microorganisms provided in Table 10. If your protocol requires further identification testing, you will need a manual or automated biochemical or molecular system.



3.2 Medium Information

In Table 2, record the lot number and expiration date of the agar used for growing the microorganisms.

Certificates of Analysis for each medium should be attached to the Verification paper work. Check (✔) if Certificate of Analysis is attached. Table 2: Media used for Verification

Medium Manufacturer Catalog Number

Lot Number

Expiry Date Certificate of Analysis (✔)

Recorded By

Sabouraud Dextrose Agar

Soybean Casein Agar (Tryptic Soy

Agar)

Columbia Blood Agar

3.3 Documentation of Traceability to Reference Culture In Table 3, record the lot number and the expiration date of each microorganism strain tested. Record the Hydrating Fluid lot number and the expiry date. Check the appropriate column after the Certificates of Assay and Analysis have been attached to the Verification paperwork. Both certificates document traceability to the original reference culture and the passage number from reference culture. Table 3: Documentation of Microorganism and Hydrating Fluid Lot Numbers

Microorganism Catalog No.

Microorganism Lot Number

Expiry Date

Hydrating Fluid Lot Number

Expiry Date

Certificate of

Assay (✔)

Certificate of

Analysis (✔)

Recorded by

Aspergillus brasiliensis

0392A

Bacillus subtilis subsp. spizizenii

0486A

Candida albicans 0443A

Clostridium sporogenes

0317A


0487A

Escherichia coli 0483A

Pseudomonas aeruginosa

0484A

Staphylococcus aureus subsp.

aureus 0485A



3.4 Counts and Purity For each EZ‐Accu Shot™ strain, do the following:

1. Remove one vial of Hydrating Fluid and one foil pouch from the canister. Remove vial containing the lyophilized microorganism pellet from the foil pouch. Allow unopened vials to equilibrate to room temperature.

2. Label Hydrating Fluid vial with microorganism lot number. 3. Remove screw top and stopper from Hydrating Fluid. 4. Remove screw top from vial containing lyophilized pellet. 5. Tip pellet into Hydrating Fluid. 6. Vortex 5 seconds or until suspension is homogenous. 7. Dispense 0.1 mL (100 µl) of suspension per agar plate. Perform a total of five replicates. 8. Spread inoculum evenly across plate with sterile spreader. 9. Incubate agar plates. Follow directions in Table 4 for temperature and length of time for incubation.

Record incubator identity, incubator temperature range, date and time when incubation begins and initials in Table 5.

10. Recap the microorganism vial and store the suspension at 2‐8°C if intending to verify 8 hour stability. (This step is not necessary if the laboratory never plans to store the suspension.) Record refrigerator identity, refrigerator temperature range, date and time suspension is placed in the refrigerator, and initials in Table 7 in Section 3.5, Verification of Stability.

11. Record in Table 5 the date and time when incubation ends. Record initials. 12. Count the colonies on each replicate plate and record the number in Table 6. Determine and record

average count. 13. Inspect the plates for purity. If no contamination is seen, record S in the Purity Column in Table 6. If

contamination is seen, record U. Date and initial.

Table 4: Incubation Instructions EZ‐Accu Shot™ Microorganism

Product Number

Media Temperature

Range Pharmacopeia Incubation

Instructions Check Macroscopic Characteristics

Atmosphere

Aspergillus brasiliensis 0392A Soybean Casein Agar1

30‐35°C ≤5 days 4‐5 days Aerobic

Aspergillus brasiliensis 0392A Sabouraud Dextrose Agar

20‐25°C ≤5 days 4‐5 days Aerobic


0486A Soybean Casein Agar1

30‐35°C ≤3 days 24 hours Aerobic

Candida albicans 0443A Soybean Casein Agar1


Candida albicans 0443A Sabouraud Dextrose Agar


Clostridium sporogenes 0317A Columbia Blood Agar

30‐35°C 48 hours 48hours Anaerobic

Clostridium sporogenes 0487A Columbia Blood Agar

30‐35°C 48 hours 48 hours Anaerobic

Escherichia coli 0483A Soybean Casein Agar1





Staphylococcus aureus subsp. aureus



1 Soybean Casein Agar is also known as Tryptic Soy Agar



Table 5: Incubation Data

Microorganism Name Catalog Number

Incubator Identity

Incubator Temperature

Range

Date and Time Incubation Begins

By Date and Time Incubation

ends By

Aspergillus brasiliensis 0392A


0486A


Clostridium sporogenes 0317A




0484A


0485A

Table 6: Microorganism Counts and Purity

Microorganism

Catalog Number

Type of

Agar1

CFU Count

Avg. Count

Purity S/U

Date By

1 2 3 4 5

A. brasiliensis 0392A SCA

A. brasiliensis 0392A SDA

B. subtilis subsp. spizizenii

0486A SCA

C. albicans 0443A SCA

C. albicans 0443A SDA

C. sporogenes 0317A CBA

C. sporogenes 0487A CBA

E. coli 0483A SCA

P. aeruginosa 0484A SCA

S. aureus subsp. aureus

0485A SCA

1SCA = Soybean Casein Agar, SDA = Sabouraud Dextrose Agar, CBA = Columbia Blood Agar

3.5 Stability

For each EZ‐Accu Shot™ strain, do the following:

1. Remove suspension from refrigerator after 8 hours (or less if laboratory is planning for shorter storage duration). Record date and time when the suspension is taken out of the refrigerator and record initials in Table 7.



2. Remix suspension on vortex mixer. 3. Dispense 0.1 mL of suspension per plate. Set up a total of five replicates. 4. Spread inoculum evenly across plate with sterile spreader. 5. Incubate second set of agar plates at the same temperature and for the same length of time as the first

set of agar plates. Record incubator identity, incubator temperature range, date and time incubation begins, and initials in Table 8.

6. Record the date and time when incubation ends and initials in Table 8. 7. Count the colonies on each replicate plate. Record the number in Table 9. Determine and record

average count. Record date and initials. Table 7: Refrigeration Data


Catalog Number

Refrigerator Identity

Refrigerator Temperature

Range

Date and Time Refrigeration

Begins By

Date and Time Refrigeration Ends

By



0486A



0317A


0487A



0484A


0485A

Table 8: Incubation Data for Refrigerated Suspensions


Catalog Number

Incubator Identity

Incubator Temperature

Range

Date and Time Incubation Begins

By Date and Time Incubation Ends

By

Aspergillus brasiliensis 0392A Bacillus subtilis subsp.

spizizenii 0486A



0317A


0487A



0484A


0485A



Table 9: Microorganism counts after refrigeration

Microorganism

Catalog Number

Type of Agar

CFU Count

Avg. Count

Date By

1 2 3 4 5

Aspergillus brasiliensis 0392A SCA

Aspergillus brasiliensis 0392A SDA


0486A SCA

Candida albicans 0443A SCA

Candida albicans 0443A SDA

Clostridium sporogenes 0317A CBA

Clostridium sporogenes 0487A CBA

Escherichia coli 0483A SCA


0484A SCA


0485A SCA


3.6 Macroscopic and Microscopic Characteristics

1. After incubation examine colonies from all strains macroscopically. Colony appearance should agree with descriptions listed in Table 10. Enter S (satisfactory appearance) or U (unsatisfactory appearance) in appropriate column. Record date and initials.

2. Use the Gram Stain method to stain bacterial colonies from each strain. Examine all strains microscopically. Microscopic appearance should agree with descriptions listed in Table 10. Enter S (satisfactory appearance) or U (unsatisfactory appearance) in appropriate column. Record date and initials.

3. Examine Aspergillus brasiliensis microscopically after spores have formed (this may take 5‐7 days). To do this, firmly press the sticky side of transparent tape to the surface of the fungal culture. Gently place the tape on a drop of Lactophenol Blue on a clear glass slide. Examine slide microscopically (a magnification of 400X works well). Microscopic appearance should agree with descriptions listed in Table 10. Enter S (satisfactory appearance) or U (unsatisfactory appearance) in appropriate column. Record date and initials.

4. If SOP calls for further testing, subculture colonies from each strain onto fresh agar plates (the new plates are the fifth passage from Reference Culture).

5. After incubation, perform identification tests, if required to do so by SOP. Describe identification method in Section 3.7.

6. Record identification in Table 11. Enter S (satisfactory results) or U (unsatisfactory results) in appropriate column. Record date and initials. Attach worksheets and results.

7. Autoclave contaminated articles or dispose of them in a biohazard container.



Table 10: Macroscopic and Microscopic Characteristics

Strain Cat. # Agar1 Macroscopic description S/U Date By Microscopic description S/U Date By

A. brasiliensis

0392A SCA,

30‐35°C, 4‐5 days.

By 4 days, rapidly growing colonies which are initially

white or pale yellow, quickly become black with

conidia (spore) production. Reverse is

pale yellow.

Lactophenol Blue Stain: chains of small conidia which arise from short sterigmata arranged radially over the surface of the vesicle.

A. brasiliensis

0392A SDA,

20‐25°C, 4‐5 days.

By 4 days, rapidly growing white colonies which

quickly become black with conidia (spore) production.

Lactophenol Blue Stain: chains of small conidia which arise from short sterigmata arranged radially over the surface of the vesicle.

B.subtilis subsp. spizizenii

0486A SCA,

30‐35°C, 24 hours.

Large, irregular, flat, undulate edge, cream and wrinkled with ground glass appearance; slight yellow coloring may appear in wrinkles by 48 hours.

Straight, gram positive rod, with an ellipsoidal, central, or terminal endospore.

C. albicans 0443A SCA,

30‐35°C, 48 hours.

Large, white, circular, convex, dull colonies.

Gram positive, spherical,

budding yeast cells.

C. albicans 0443A SDA,

20‐25°C, 48 hours.

Large, white, circular, convex, dull colonies.

Gram positive, ovoid, budding yeast cells.

C. sporogenes

0317A

CBA, anaerobic conditions, 30‐35°C, 48 hours.

Large, irregularly circular, with coarse rhizoid edge, slightly raised center, gray, beta hemolytic, swarms,

adheres to agar.

Gram positive straight rods. Spores, when seen, are oval, central or subterminal, and distend the cell slightly.

C. sporogenes

0487A

CBA, anaerobic conditions, 30‐35°C, 48 hours.

Large, irregularly circular, with coarse rhizoid edge, slightly raised center, gray, beta hemolytic, swarms,

adheres to agar.

Gram positive straight rods. Spores, when seen, are oval, central or subterminal, and distend the cell slightly.

E. coli 0483A SCA,

30‐35°C, 24 hours.

Medium to large, creamy, low convex, slightly rough,

slightly erose. Gram negative straight rod.

P. aeruginosa

0484A SCA,

30‐35°C, 24 hours.

Medium to large, flat, circular to irregular shaped, cream.

Straight or slightly curved

gram negative rod.

S. aureus subsp. aureus

0485A SCA,

30‐35°C, 24 hours.

Medium, convex, circular, glistening, smooth,

creamy, opaque, light gold colonies.

Gram positive cocci

occurring singly, in pairs, and in irregular clusters.




3.7 Biochemical and/or Molecular Identity (If Required)

Table 11: Identification Test Results

Microorganism Catalog Number

Biochemical/Molecular Identification S/U Date By



0486A






0484A


0485A

4. Summary and Conclusion Determine if each EZ‐Accu Shot™ lyophilized microorganism strain meets acceptance criteria.

4.1 Summary of Data Enter S (Satisfactory) or U (Unsatisfactory) for each category. Date and initial.



Table 12: Summary

Microorganism Cat. no. Type of media1

Traceability S/U

CFU count S/U

8 hour Stability S/U

Purity S/U

Identity S/U

Date By


0392A SCA


0392A SDA


0486A SCA

Candida albicans 0443A SCA

Candida albicans 0443A SDA


0317A CBA


0487A CBA

Escherichia coli 0483A SCA


0484A SCA

Staphylococcus aureus subsp.

aureus

0485A SCA

1SCA = Soybean Casein Agar, SDA = Sabouraud Dextrose Agar, CBA = Columbia Blood Agar 4.2 Comments By _______________________________________________ Date ___________________________________



4.3 Conclusion By _______________________________________________ Date __________________________________

4.4 Review and Approval

Name Title Signature Date

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